In Vitro Drug Metabolism Using Liver Microsomes.

Knowledge of the metabolic stability of newly discovered drug candidates eliminated by metabolism is essential for predicting the pharmacokinetic (PK) parameters that underpin dosing and dosage frequency. Further, characterization of the enzyme(s) responsible for metabolism (reaction phenotyping) allows prediction, at least at the qualitative level, of factors (including metabolic drug-drug interactions) likely to alter the clearance of both new chemical entities (NCEs) and established drugs. Microsomes are typically used as the enzyme source for the measurement of metabolic stability and for reaction phenotyping because they express the major drug-metabolizing enzymes cytochrome P450 (CYP) and UDP-glucuronosyltransferase (UGT), along with others that contribute to drug metabolism. Described in this unit are methods for microsome isolation, as well as for the determination of metabolic stability and metabolite formation (including kinetics). © 2016 by John Wiley & Sons, Inc.

A multi-endpoint evaluation of cytochrome P450 1A2, 2B6 and 3A4 induction response in human hepatocyte cultures after treatment with ß-naphthoflavone, phenobarbital and rifampicin.

U.S. FDA and EMEA guidance recommend that the preferred in vitro model for cytochrome P450 induction testing is human hepatocytes coupled with acceptable inducers as controls. However, there are surprisingly few published studies characterizing this model system for dose and time-dependence response to model inducing compounds. The concentration-dependent response and time-course for the induction of CYP1A2, CYP2B6 and CYP3A4 by inducing agents ß-naphthoflavone, phenobarbital and rifampicin, respectively were examined in two or more donors using multiple end-points (mRNA, enzyme activity and Western blot analysis). Depending on the endpoint, exposure time for maximal response of CYP induction potential for the three enzymes ranged from 24 to 72 hours. Of the concentrations of BNF, PB and RIF tested, those which gave the maximal response were found to be 33 µM, > 2 mM and 10 µM, respectively.

Differential time- and NADPH-dependent inhibition of CYP2C19 by enantiomers of fluoxetine.

Fluoxetine [+/--N-methyl-3-phenyl-3-[(alpha, alpha, (-trifluoro-p-tolyl)oxy]-propylamine)] a selective serotonin reuptake inhibitor, is widely used in treating depression and other serotonin-dependent disease conditions. Racemic, (R)- and (S)-fluoxetine are potent reversible inhibitors of CYP2D6, and the racemate has been shown to be a mechanism-based inhibitor of CYP3A4. Racemic fluoxetine also demonstrates time- and concentration-dependent inhibition of CYP2C19 catalytic activity in vitro. In this study, we compared fluoxetine, its (R)- and (S)-enantiomers, ticlopidine, and S-benzylnirvanol as potential time-dependent inhibitors of human liver microsomal CYP2C19. In a reversible inhibition protocol (30 min preincubation with liver microsomes without NADPH), we found (R)-, (S)- and racemic fluoxetine to be moderate inhibitors with IC(50) values of 21, 93, and 27 microM, respectively. However, when the preincubation was supplemented with NADPH, IC(50) values shifted to 4.0, 3.4, and 3.0 microM, respectively resulting in IC(50) shifts of 5.2-, 28-, and 9.3-fold. Ticlopidine showed a 1.8-fold shift in IC(50) value, and S-benzylnirvanol shifted right (0.41-fold shift). Follow-up K(I) and k(inact) determinations with fluoxetine confirmed time-dependent inhibition [K(I) values of 6.5, 47, and 14 microM; k(inact) values of 0.023, 0.085, 0.030 min(-1) for (R)-, (S)-, and racemate, respectively]. Although the (S)-isomer exhibits a much lower affinity for CYP2C19 inactivation relative to the (R)-enantiomer, it exhibits a more rapid rate of inactivation. Racemic norfluoxetine exhibited an 11-fold shift (18-1.5 microM) in IC(50) value, suggesting that conversion of fluoxetine to this metabolite represents a metabolic pathway leading to time-dependent inhibition. These data provide an improved understanding of the drug-interaction potential of fluoxetine.

A novel design of artificial membrane for improving the PAMPA model.

PURPOSE: Since the first demonstration of PAMPA, the artificial membrane has been traditionally prepared by impregnating a porous filter with a solution of lipid mixture. While the lipid solution-based method is simple and seems to provide good predictability for many compounds, it is challenged by several shortcomings including reproducibility, stability, mass retention and the incorrect prediction of a group of highly permeable compounds including caffeine and antipyrine. Here we present the validation of a novel artificial membrane formed by constructing a lipid/oil/lipid tri-layer in the porous filter. METHODS: Permeability values obtained from traditional and new artificial membrane were compared for their correlation with Caco-2 and human absorption values. Mass retention, stability and organic solvent compatibility of the new artificial membrane were studied. RESULTS: The new artificial membrane correctly predicts the permeability of the traditionally under-predicted compounds and improves the correlation with Caco-2 and human absorption values. Furthermore, the new artificial membrane reduces the mass retention of compounds that are highly retained by the traditional artificial membrane. The new artificial membrane is also found to be robust enough to sustain long term storage and has good compatibility with organic solvents. CONCLUSIONS: The new artificial membrane provides an improved PAMPA model.

Comparison of species differences of P-glycoproteins in beagle dog, rhesus monkey, and human using Atpase activity assays.

P-glycoprotein (P-gp) is a transmembrane efflux transporter which possesses many important functions in drug absorption, disposition, metabolism, and toxicity. The ultimate goal of investigating drug interactions between P-gp and drug molecules in early drug discovery is to understand the contribution of P-gp to the pharmacokinetic and pharmacodynamic properties of drug candidates and to project drug-drug interaction (DDI) potentials in humans. Understanding species differences in P-gp activities further helps the prediction of P-gp-mediated drug disposition and DDI in humans from preclinical pharmacokinetics data. The objective of the present study is to investigate the species difference in P-gp activities, via P-gp ATPase assays, using rhesus monkey Mdr1, beagle dog Mdr1, and human MDR1 expressed insect cell membranes. Twenty-one compounds with diverse chemical structures and different P-gp binding sites were chosen for the ATPase assays. P-gp ATPase binding affinities (alphaKa) and fold increases in P-gp ATPase activities (beta) of P-gp substrates were determined. Consistent with the gene and amino acid similarity, the binding affinities of test compounds to rhesus monkey P-gp were much closer to those of human P-gp than beagle dog P-gp. This is the first study which investigates the ligand affinities of P-gp from three different species. The result of this study provides an example of how to use membrane P-gp ATPase assays to evaluate interspecies P-gp differences.

Determination of CYP2B6 component of 7-ethoxy-4-trifluoromethylcoumarin O-deethylation activity in human liver microsomes.

The cytochrome P450 enzyme CYP2B6 plays an important role in the metabolism of structurally diverse drugs, including the anticancer drug cyclophosphamide, and may be an important determinant of clinical responses to these agents. A spectrofluorometric method is described for the determination of CYP2B6-catalyzed 7-ethoxy-4-trifluoromethylcoumarin O-deethylation activity in human liver microsomes. The specificity of this method for CYP2B6 is increased by the use of inhibitory antibodies to CYP1A2, CYP2C, and CYP2E1, which block the contributions of these higher-K(m) enzymes to human liver microsomal metabolism of 7-ethoxy-4-trifluoromethylcoumarin. The enzymatic product, 7-hydroxy-4-trifluoromethylcoumarin, is monitored by fluorescence using an excitation wavelength of 410 nm and an emission wavelength of 510 nm. This approach can be modified to assay the catalytic activity of cDNA-expressed CYP2B6.

High-performance liquid chromatography analysis of CYP2C8-catalyzed paclitaxel 6alpha-hydroxylation.

Paclitaxel (Taxol) is a naturally occurring member of the taxane family of antitumor drugs, which act by stabilizing microtubules. Paclitaxel is inactivated in human liver by a cytochrome P450 (P450)-catalyzed 6alpha-hydroxylation reaction. A reverse-phase, high-performance liquid chromatographic assay is described for the analysis of paclitaxel 6alpha-hydroxylation catalyzed by human liver microsomes or cDNA-expressed P450 enzyme CYP2C8. Analytical separations are achieved using a C18 column with a linear gradient of 10-100% methanol, with detection at 230 nm. This method is applicable to enzymatic studies for determination of CYP2C8-catalyzed paclitaxel 6alpha-hydroxylation activity.

Determination of CYP2C9-catalyzed diclofenac 4'-hydroxylation by high-performance liquid chromatography.

A reverse-phase, high-performance liquid chromatography method is described for quantification of diclofenac 4'-hydroxylation catalyzed by human liver microsomes or cDNA-expressed CYP2C9. Analytical separation is achieved using a C18 column developed with a gradient of 30% acetonitrile and 2 mM perchloric acid in water to 100% methanol, with detection at 280 nm. This method is applicable to enzymatic studies for determination of CYP2C9-catalyzed diclofenac 4'-hydroxylation activity.

CYP2C19-mediated (S)-mephenytoin 4'-hydroxylation assayed by high-performance liquid chromatography with radiometric detection.

A reverse-phase, high-performance liquid chromatography method is described for the quantification of 4'-hydroxymephenytoin formed enzymatically from 14C-labeled (S)-mephenytoin following incubation with cDNA-expressed CYP2C19 or human liver microsomes. Analytical separation is achieved using a C18 column developed with a gradient from 10 to 100% methanol, with detection using a scintillation detector. This method is applicable to enzymatic studies for determination of CYP2C19-catalyzed (S)-mephenytoin 4'-hydroxylation activity.

CYP2D6-dependent bufuralol 1'-hydroxylation assayed by reverse-phase ion-pair high-performance liquid chromatography with fluorescence detection.

A reverse-phase, high-performance liquid chromatography method is described for the quantification of 1'-hydroxybufuralol formed enzymatically by the incubation of bufuralol with cDNA-expressed CYP2D6 or human liver microsomes. Analytical separation is achieved using a C18 column and a mobile phase consisting of 30% acetonitrile and 2 mM perchloric acid, with detection by fluorescence using an excitation wavelength of 252 nm and an emission wavelength of 302 nm. This method is applicable to enzymatic studies for determination of CYP2D6-catalyzed bufuralol 1'-hydroxylation activity.

Spectrophotometric analysis of human CYP2E1-catalyzed p-nitrophenol hydroxylation.

The cytochrome P450 enzyme CYP2E1 catalyzes the oxidative metabolism of many solvents and other small organic molecules. A spectrophotometric method is described for determination of CYP2E1 activity by monitoring the formation of p-nitrocatechol from p-nitrophenol by cDNA-expressed CYP2E1 or isolated liver microsomes. The enzymatic product, p-nitrocatechol, is assayed at 535 nm after acidification of the reaction mixture with trichloroacetic acid followed by neutralization using 2 M NaOH. This method is applicable to enzymatic studies for determination of P450-catalyzed p-nitrophenol hydroxylation activity.

Determination of CYP4A11-catalyzed lauric acid 12-hydroxylation by high-performance liquid chromatography with radiometric detection.

Lauric acid serves as an endogenous substrate for the cytochrome P450 enzyme CYP4A11. A reverse-phase, high-performance liquid chromatography method is described for the quantification of 12-hydroxylauric acid formed enzymatically by incubation of 14C-labeled lauric acid with cDNA-expressed CYP4A11 or human liver microsomes. Analytical separation is achieved using a C18 column and a gradient of 30% acetonitrile and 2 mM perchloric acid to 100% methanol, using a detection scintillation counter. This method is applicable to enzymatic studies for determination of lauric acid 12-hydroxylation activity.

Human cytochrome p450 induction and inhibition potential of clevidipine and its primary metabolite h152/81.

Clevidipine is a short-acting dihydropyridine calcium channel antagonist under development for treatment of perioperative hypertension. Patients treated with clevidipine are likely to be comedicated. Therefore, the potential for clevidipine and its major metabolite H152/81 to elicit drug interactions by induction or inhibition of cytochrome P450 was investigated. Induction of CYP1A2, CYP2C9, and CYP3A4 was examined in primary human hepatocytes treated with clevidipine at 1, 10, and 100 microM. Clevidipine was found to be an inducer of CYP3A4, but not of CYP1A2 or CYP2C9, at the 10 microM and 100 microM concentrations of clevidipine tested. Induction response for CYP3A4 to 100 microM clevidipine was approximately 20% of that of the positive control inducer rifampicin. The response of H152/81 was similar. Using cDNA-expressed enzymes, clevidipine inhibited CYP2C9, CYP2C19, and CYP3A4 activities with IC(50) values below 10 microM, whereas CYP1A2, CYP2D6, and CYP2E1 activities were not substantially inhibited (IC(50) values >70 microM). The K(i) values for CYP2C9 and CYP2C19 were 1.7 and 3.3 microM, respectively, and those for CYP3A4 were 8.3 and 2.9 microM, using two substrates, testosterone and midazolam, respectively. These values are at least 10 times higher than the highest clevidipine concentration typically seen in the clinic. Little or no inhibition by H152/81 was found for the enzyme activities mentioned above (IC(50) values >or= 69 microM). The present study demonstrates that it is highly unlikely for clevidipine or its major metabolite to cause cytochrome P450-related drug interactions when used in the dose range required to manage hypertension in humans.

High-Performance Liquid Chromatography Analysis of CYP2C8-Catalyzed Paclitaxel 6α-Hydroxylation.

Paclitaxel (Taxol) is a naturally occurring member of the taxane family of antitumor drugs, which act by stabilizing microtubules. Paclitaxel is inactivated in human liver by a cytochrome P450 (P450)-catalyzed 6α-hydroxylation reaction. A reverse-phase, high-performance liquid chromatographic assay is described for the analysis of paclitaxel 6α-hydroxylation catalyzed by human liver microsomes or cDNA-expressed P450 enzyme CYP2C8. Analytical separations are achieved using a C18 column with a linear gradient of 10-100% methanol, with detection at 230 nm. This method is applicable to enzymatic studies for determination of CYP2C8-catalyzed paclitaxel 6α-hydroxylation activity.

Human-cell mutagens in respirable airborne particles in the northeastern United States. 1. Mutagenicity of fractionated samples.

Few studies have characterized the regional scale (300-500 km) variability of the mutagenicity of respirable airborne particles (PM2.5). We previously collected 24-h PM2.5 samples for 1 year from background, suburban, and urban sites in Massachusetts (MA) and rural and urban sites in upstate New York (NY) (n = 53-60 samples per site). Bimonthly composites of these samples were mutagenic to human cells. The present report describes our effort to identify chemical classes responsible for the mutagenicity of the samples, to quantify spatial differences in mutagenicity, and to compare the mutagenicity of samples composited in different ways. Organic extracts and HPLC fractions (two nonpolar, one semipolar, and one polar) of annual composites were tested for mutagenicity in the h1A1v2 cells, a line of human B-lymphoblastoid cells that express cytochrome P450 CYP1A1 cDNA. The mutagenic potency (induced mutant fraction per microg organic carbon) of the semipolarfractions was the highest at all five sites, accounting for 35-82% of total mutagenic potency of the samples, vs the nonpolar (4-38%) and polar (14-32%) fractions. These results are consistent with previous studies. While unfractionated extracts exhibited no spatial variations, the mutagenicity of semipolar fractions at the NY sites was approximately 2-fold higher than at the MA sites. This suggests there may be significant regional differences in the sources and/ or transport and transformation of mutagenic compounds in PM2.5. In addition, mutagenic potency was sensitive to whether samples were fractionated and how they were composited: unfractionated annual composite samples at the NY sites were significantly less mutagenic than their semipolar fractions and the annual average of bimonthly composites; spatial differences in the mutagenic potency of bimonthly composites and the semipolar fractions were not apparent in the annual composites.

Highly selective inhibition of human CYP3Aa in vitro by azamulin and evidence that inhibition is irreversible.

Azamulin [14-O-(5-(2-amino-1,3,4-triazolyl)thioacetyl)-dihydromutilin] is an azole derivative of the pleuromutilin class of antiinfectives. We tested the inhibition potency of azamulin toward 18 cytochromes P450 using human liver microsomes or microsomes from insect cells expressing single isoforms. In a competitive inhibition model, IC(50) values for CYP3A (0.03-0.24 microM) were at least 100-fold lower than all other non-CYP3A enzymes except CYP2J2 ( approximately 50-fold lower). The IC(50) value with heterologously expressed CYP3A4 was 15-fold and 13-fold less than those of CYP3A5 and CYP3A7, respectively. The reference inhibitor ketoconazole was less selective and exhibited potent inhibition (IC(50) values <10 microM) for CYP1A1, CYP1B1, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP4F2, and CYP4F12. Inhibition of CYP3A by azamulin appeared sigmoidal and well behaved with the substrates 7-benzyloxy-4-trifluoromethylcoumarin, testosterone, and midazolam. Preincubation of 4.8 microM azamulin in the presence of NADPH for 10 min inhibited approximately 95% of testosterone 6beta-hydroxylase activity compared with preincubation in the absence of NADPH. Catalytic activities of CYP1A2, CYP2C8, CYP2C9, CYP2C19, CYP2D6, and CYP2E1 were unaffected by similar experiments. Incubation of azamulin with heterologously expressed CYP3A4 yielded a type I binding spectrum with a spectral dissociation constant of 3.5 microM, whereas no interaction was found with CYP2D6. Azamulin exhibited good chemical stability when stored in acetonitrile for up to 12 days. Aqueous solubility was found to be >300 microM. Azamulin represents an important new chemical tool for use in characterizing the contribution of CYP3A to the metabolism of xenobiotics.

Assessing pregnancy risks of azole antifungals using a high throughput aromatase inhibition assay.

UNLABELLED: Human aromatase (CYP19) converts C19 androgens to aromatic C18 estrogenic steroids. Its activity is critical for early and mid pregnancy maintenance and in regulating parturition in late pregnancy. Past studies have utilized placental microsome tritiated water release assay to assess drug-hormone interactions with estrogen synthesis. We compared data from human placental assays with BD Gentest's high throughput recombinant CYP19 enzyme assay using the fluorometric substrate dibenzylfluorescein. We tested a panel of azole antifungal agents that are commonly administered to women of childbearing potential, for their potential to inhibit aromatase. Potency varied by several orders of magnitude. Plasma and tissue levels of some azole drugs following oral or topical administration are at or above these IC50 values. These include the oral agents fluconazole and ketoconazole, and the topical agents econazole, bifonazole, clotrimazole, miconazole, and sulconazole. CONCLUSIONS: 1. Recombinant enzyme assay data are comparable to the human placental assay data in both SAR rank order and potency. 2. Plasma and tissue levels of some azole drugs following oral or topical administration are at or above these IC50 values. Therefore, some azole drugs may disrupt estrogen production in pregnancy, affecting pregnancy outcome. 3. Recombinant CYP19 assay using the fluorometric substrate dibenzylfluorescein, demonstrates rapid screening potential for chemicals that may affect pregnancy outcome as a result of CYP19 inhibition.

CYP2C8 polymorphisms in Caucasians and their relationship with paclitaxel 6alpha-hydroxylase activity in human liver microsomes.

Published cDNA sequences suggest the existence of non-synonymous single nucleotide polymorphisms in the cytochrome P450 CYP2C8. To determine whether these polymorphisms could be confirmed in a Caucasian population and to investigate whether additional polymorphisms occur in the coding and upstream regions of this gene, we screened for previously described and for novel polymorphisms using PCR-RFLP and SSCP analysis. We confirmed the existence of two of the previously detected polymorphisms which give rise to the amino acid substitutions I264M and K399R, respectively, but failed to detect three others in our population. We also confirmed that a recently identified polymorphism (R139K) is linked to K399R (CYP2C8*3) in our study population. The allele frequencies for the I264M (CYP2C8*4 allele) and the CYP2C8*3 allele were 0.075 and 0.15, respectively. Three novel polymorphisms (T-370G, C-271A and T1196C/L390S) were also detected with the upstream polymorphisms showing allele frequencies of 0.061 and 0.196, respectively, but the L390S polymorphism detected only in a single subject. An additional single subject was heterozygous for a polymorphism recently described in African-Americans (A805T; CYP2C8*2 allele). The functional significance of the two upstream polymorphisms and the CYP2C8*3 and CYP2C8*4 alleles was investigated in human liver microsomes. Samples heterozygous for CYP2C8*3 showed significantly lower paclitaxel 6alpha-hydroxylase activity compared with wild-type samples. Median activity associated with CYP2C8*4 also appeared lower than the wild-type but the difference was not significant. There was no evidence that either upstream polymorphism gave rise to altered CYP2C8 expression.